The objective of the proposed project is to use the EcoRI restriction and modification enzymes as model systems for study of sequence-specific DNA-protein interaction. The specific aim is to gain insight into the molecular basis of Type II host restriction and modification as typified by the EcoRI enzymes. Homogeneous EcoRI endonuclease and methylase will be subjected to extensive physical and chemical analysis. The goal of these studies will be to elucidate the catalytically active oligomeric states of the proteins and to identify possible regions of primary sequence homology between endonuclease and methylase. Catalytic properties of the pure enzymes will be extensively studied. This phase of the research will include bisubstrate kinetic analysis of the methylase reaction; investigation of the possibility of processive or random mechanisms; and in order to identify DNA determinants important in recognition, assessment of catalytic activity on DNA containing nucleotide analogs selectively substituted in the RI site. We will investigate binding of EcoRI enzymes to natural DNA molecules and to short DNA fragments containing a single RI site, with the aim being to obtain thermodynamic and kinetic parameters characteristic of interaction of these proteins with DNA. By chemical and mutational modification of the proteins, we will attempt to identify amino acid residues important in sequence recognition and catalysis.